The present invention is related to the field of molecular biology, and more particular, to polynucleotide synthesis. The present invention also relates to a substantially pure thermostable exonuclease, the cloning and expression of a thermostable exonuclease III in E. coli, and its use in amplification reactions. The invention facilitates the high fidelity amplification of DNA under conditions which allow decontamination from carry over and the synthesis of long products. The invention may be used for a variety of industrial, medical and forensic purposes.
In vitro nucleic acid synthesis is routinely performed with DNA polymerases with or without additional polypeptides. DNA polymerases are a family of enzymes involved in DNA replication and repair. Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli. See, for example, Bessman et al. (1957) J. Biol. Chem. 223:171-177, and Buttin and Komberg, (1966) J. Biol. Chem. 241:5419-5427.
Research has also been conducted on the isolation and purification of DNA polymerases from thermophiles, such as Thermus aquaticus. Chien, A. et al., (1976) J. Bacteriol. 127:1550-1557 discloses the isolation and purification of a DNA polymerase with a temperature optimum of 80° C. from Thermus aquaticus YT1 strain. U.S. Pat. No. 4,889,818 discloses a purified thermostable DNA polymerase from T. aquaticus, Taq polymerase, having a molecular weight of about 86,000 to 90,000 daltons. In addition, European Patent Application 0 258 017 discloses Taq polymerase as the preferred enzyme for use in the PCR process.
Research has indicated that while Taq DNA polymerase has a 5′-3′ polymerase-dependent exonuclease function, Taq DNA polymerase does not possess a 3′-5′ exonuclease III function (Lawyer, F. C. et al., (1989) J. Biol. Chem., 264:6427-6437; Bemad A., et al. (1989) Cell 59:219). The 3′-5′ exonuclease activity of DNA polymerases is commonly referred to as “proofreading activity”. The 3′-5′ exonuclease activity removes bases which are mismatched at the 3′ end of a primer-template duplex. The presence of 3′-5′ exonuclease activity may be advantageous as it leads to an increase in fidelity of replication of nucleic acid strands and to the elongation of prematurely terminated products. As Taq DNA polymerase is not able to remove mismatched primer ends it is prone to base incorporation errors, making its use in certain applications undesirable. For example, attempting to clone an amplified gene is problematic since any one copy of the gene may contain an error due to a random misincorporation event. Depending on the cycle in which that error occurs (e.g., in an early replication cycle), the entire DNA amplified could contain the erroneously incorporated base, thus, giving rise to a mutated gene product.
There are several thermostable DNA polymerases known in the art which exhibit 3′-5′ exonuclease activity, like B-type polymerases from thermophilic Archaebacteria which are used for high fidelity DNA amplification. Thermostable polymerases exhibiting 3′-5′ exonuclease activity may be isolated or cloned from Pyrococcus (Purified thermostable Pyrococcus furiosus DNA polymerase, Mathur E., Stratagene, WO 92/09689, U.S. Pat. No. 5,545,552; Purified thermostable DNA polymerase from Pyrococcus species, Comb D. G. et al., New England Biolabs, Inc., EP 0 547 359; Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus, Uemori T. et al. (1993) Nucl. Acids Res., 21:259-265.), from Pyrodictium spec. (Thermostable nucleic acid polymerase, Gelfand D. H., F. Hoffmann-La Roche AG, EP 0 624 641; Purified thermostable nucleic acid polymerase and DNA coding sequences from Pyrodictium species, Gelfand D. H., Hoffmann-La Roche Inc., U.S. Pat. No. 5,491,086), from Thermococcus (e.g. Thermostable DNA polymerase from Thermococcus spec. TY, Niehaus F., et al. WO 97/35988; Purified Thermocccus barossii DNA polymerase, Luhm R. A., Pharmacia Biotech, Inc., WO 96/22389; DNA polymerase from Thermococcus barossii with intermediate exonuclease activity and better long term stability at high temperature, useful for DNA sequencing, PCR etc., Dhennezel O. B., Pharmacia Biotech Inc., WO 96/22389; A purified thermostable DNA polymerase from Thermococcus litoralis for use in DNA manipulations, Comb D. G., New England Biolabs, Inc., U.S. Pat. No. 5,322,785, EP 0 455 430; Recombinant thermostable DNA polymerase from Archaebacteria, Comb D. G., New England Biolabs, Inc., U.S. Pat. No. 5,352,778, EP 0 547 920, EP 0 701 000; New isolated thermostable DNA polymerase obtained from Thermococcus gorgonarius, Angerer B. et al. Boehringer Mannheim GmbH, WO 98/14590.
Another possibility of conferring PCR in the presence of a proofreading function is the use of a mixture of polymerase enzymes, one polymerase exhibiting such a proofreading activity. (e.g. Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of primer extension, Barnes W. M., U.S. Pat. No. 5,436,149, EP 0 693 078; Novel polymerase compositions and uses thereof, Sorge J. A., Stratagene, WO 95/16028). It is common practice to use a formulation of a thermostable DNA polymerase comprising a majority component of at least one thermostable DNA polymerase which lacks 3′-5′ exonuclease activity and a minority component exhibiting 3′-5′ exonuclease activity e.g. Taq polymerase and Pfu DNA polymerase. In these mixtures the processivity is conferred by the pol I-type enzyme like Taq polymerase, the proofreading function by the thermostable B-type polymerase like Pfu. High fidelity DNA synthesis is one desirable parameter in nucleic acid amplification, another important feature is the possibility of decontamination.
The polymerase chain reaction can amplify a single molecule over a billionfold. Thus, even minuscule amounts of a contaminant can be amplified and lead to a false positive result. Such contaminants are often poducts from previous PCR amplifications (carry-over contamination). Therefore, researchers have developed methods to avoid such a contamination.
The procedure relies on substituting dUTP for TTP during PCR amplification to produce uracil-containing DNA (U-DNA). Treating subsequent PCR reaction mixtures with Uracil-DNA-Glycosylase (UNG) prior to PCR amplification the contaminating nucleic acid is degraded and not suitable for amplification. dUTP can be readily incorporated by poll-type thermostable polymerases but not B-type polymerases (G. Slupphaug, et al. (1993) Anal. Biochem. 211:164-169) Low incorporation of dUTP by B-type polymerases limits their use in laboratories where the same type of template is repeatedly analyzed by PCR amplification.
Thermostable DNA polymerases exhibiting 3′-5′ exonuclease activity were also isolated from eubacterial strains like Thermotoga (Thermophilic DNA polymerases from Thermotoga neapolitana, Slater M. R. et al. Promega Corporation, WO 96/41014; Cloned DNA polymerases from Thermotoga neapolitana and mutants thereof, Hughes A. J. et al., Life Technologies, Inc. WO 96/10640; Purified thermostable nucleic acid polymerase enzyme from Termotoga maritima, Gelfand D. H. et al., CETUS Corporation, WO 92/03556) These enzymes have a strong 3′-5′ exonuclease activity which is able to eliminate misincorporated or mismatched bases. A genetically engineered version of this enzyme is commercially available as ULTma, a DNA polymerase which can be used without additional polypeptides for the PCR process. This enzyme is able to remove misincorporated bases, incorporate dUTP, but the fidelity is for unknown reasons not higher than that of Taq polymerase (Accuracy of replication in the polymerase chain reaction. Diaz R. S. et al. Braz. J. Med. Biol. Res. (1998) 31: 1239-1242; PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases, Cline J. et al., Nucleic Acids Res. (1996) 24:3546-3551).
For high fidelity DNA synthesis another alternative to the use of B-type polymerases or mixtures containing them is the use of thermophilic DNA polymerase III holoenzyme, a complex of 18 polypeptide chains. These complexes are identical to the bacterial chromosomal replicases, comprising all the factors necessary to synthesize a DNA strand of several hundred kilobases or whole chromosomes. The 10 different subunits of this enzyme, some of which are present in multiple copies, can be produced by recombinant techniques, reconstituted and used for in vitro DNA synthesis. As a possible use of these complexes PCR amplification of nucleic acis of several thousand to hundreds of thousand base pairs is proposed. (Enzyme derived from thermophilic organisms that functions as a chromosomal replicase, and preparation and uses thereof, Yurieva O. et al., The Rockefeller University, WO 98/45452; Novel thermophilic polymerase III holoenzyme, McHenry C., ENZYCO Inc., WO 99/13060)
It was aimed according to this invention to develop a high fidelity PCR system which is preferably concomitantly able to incorporate dUTP. According to the present invention a thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity is provided whereas this enzyme enhances fidelity of an amplification process when added to a second enzyme exhibiting polymerase activity. The enzyme provided can excise mismatched primer ends to allow the second enzyme exhibiting polymerase activity as e.g. Taq polymerase to reassociate and to reassume elongation during a process of synthezising DNA. The inventive enzyme is able to cooperate as proofreading enzyme with a second enzyme exhibiting polymerase activity. The enzyme that was found to be suitable for this task is e.g. a thermostable exonuclease III. Preferred is an exonuclease III working from the 3′ to 5′ direction, cleaving 5′ of the phosphate leaving 3′ hydroxyl groups and ideally working on double stranded DNA only. The 3′-5′ exonuclease functions of DNA polymerases are active on double and single stranded DNA. The latter activity may lead to primer degradation, which is undesired in PCR assays. It is preferred that the enzyme is active at 70° C. to 80° C., stable enough to survive the denaturation cycles and inactive at lower temperatures to leave the PCR products undegraded after completion of the PCR process. Enzymes exhibiting these features can be derived from thermophilic eubacteria or related enzymes from thermophilic archaea. Genomes of three thermostable archaebacteria are sequenced, Methanococcus jannaschii (Complete Genome Sequence of the Methanogenic Archaeon, Methanococcus jannaschii, Bult C. J. et al., (1996) Science 273: 1058-1072), Methanobacterium thermoautotrophicum (Complete genomic sequence of Methanobacterium thermoautotrophicum ΔH: Functional Analysis and Comparative Genomics, Smith D. R. et al., J of Bacteriology (1997) 179: 7135-7155) and Archaeoglobus fulgidus (The complete genome sequence of the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus, Klenk H.-P. et al. (1997) Nature 390: 364-370).
In particular, there is provided a thermostable enzyme obtainable from Archaeoglobus fulgidus, which catalyzes the degradation of mismatched ends of primers or polynucleotides in the 3′ to 5′ direction in double stranded DNA. The gene encoding the thermostable exonuclease III obtainable from Archaeoglobus fulgidus (Afu) was cloned, expressed in E. coli and isolated. The enzyme is active under the incubation and temperature conditions used in PCR reactions. The enzyme supports DNA polymerases like Taq in performing DNA synthesis at low error rates and synthesis of products of more than 3 kb on genomic DNA—the upper range of products synthesized by Taq polymerase—in good yields with or without dUTP present in the reaction mixture. Preferably, 50-500 ng of the exonuclease III obtainable from Afu were used per 2,5 U of Taq polymerase in order to have an optimal PCR performance. More preferably is the use of 67 ng to 380 ng of the exonuclease III obtainable from Afu per 2,5 U of the Taq polymerase in the PCR reaction.
DNA sequence (SEQ ID NO: 20) and the deduced amino acid sequence (SEQ ID NO: 21) of the gene encoding the DNA polymerase from exonuclease III Archaeoglobus fulgidis. 
Further, subject of the present invention is a composition comprising a first thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity and a second enzyme exhibiting polymerase activity whereas the fidelity of an amplification process is enhanced by the use of this composition in comparison to the use of the second enzyme alone. The inventive thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity also includes appropriate enzymes exhibiting reduced DNA polymerase activity or no such activity at all. Reduced DNA polymerase activity according to the invention means less than 50% of said activity of an enzyme exhibiting DNA polymerase activity. In a preferred embodiment the second enzyme of the inventive composition is lacking proofreading activity. In particular preferred, the second enzyme is Taq polymerase.
A further subject of the present invention is a method of DNA synthesis using a mixture comprising a first thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity and a second enzyme exhibiting polymerase activity. According to this method prematurely terminated chains are trimmed by degradation from 3′ to 5′. Mismatched ends of either a primer or the growing strand are removed according to this method.
The invention further comprises a method according to the above description whereas dUTP is present in the reaction mixture, replacing partly or completely TTP. It is preferred that according to this method uracil DNA glycosylase (UDG or UNG) is used for degradation of contaminating nucleic acids.
Preferably according to this method the mixture of a                first thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity and        a second enzyme exhibiting polymerase activity produces PCR products with lower error rates compared to PCR products produced by the second enzyme exhibiting polymerase activity in absence of the first thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity. The method in which the mixture of first thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity and a second enzyme exhibiting polymerase activity produces PCR products of greater length compared to PCR products produced by the second enzyme exhibiting polymerase activity in absence of the first thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity. Further, the first thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity is related to the Exonuclease III of E. coli, but thermostable according to this method. A further embodiment of the above described method is the method whereas PCR products with blunt ends are obtained.        
Subject of the present invention are also methods for obtaining the inventive thermostable enzyme exhibiting 3′ exonuclease-activity but essentially no DNA polymerase activity and means and materials for producing this enzyme as e.g. vectors and host cells (e.g. DSM no. 13021).
The following examples are offered for the purpose of illustrating, not limiting, the subject invention.